1. Field of the Invention
This invention pertains to the use of chemiluminescent 1,2-dioxetanes in homogenous or heterogeneous assays to detect proteases inhibitors.
2. Discussion of the Background
The identification of novel therapeutics that block or inhibit inimical proteases, or proteases that mediate disease conditions, such as the 11-kd protease encoded by the human immunodeficiency virus 1(HIV-1) is a key step in slowing the disease process of AIDS. Retroviral proteases are essential in the process of viral gag-pol polyproteins of the HIV-1 and HIV-2 viruses. There are a few highly conserved consensus sequences in retroviral polyproteins, one of which consists of a pentapeptide (Ser/Thr)-X-X'-(Tyr/Phe)-Pro (SEQ ID No:1). Cleavage occurs between the Tyr or Phe and Pro residues. Blocking activity of these proteases will interfere with the progression of HIV infection. Although potent drugs which block HIV protease activity have been found, there is an ongoing need to find and develop novel inhibitors.
Current methods utilized in rapid screening of protease inhibitors are subject to many interferences from a variety of sources. The most common non-isotopic approach is a fluorescent assay. In one case, such as in the detection of HIV protease, the fluorescent substrate is labeled with a fluorescent dansyl group on one end of a peptide and a quencher on the other. An increase in fluorescence signal occurs upon cleavage of the protease due to the fact that the emitter and the quencher are separated as described in Matayoshi et al., 1990, Science 247: 954-958. Fluorescent substrates for other proteases can be designed with a terminal fluorophore which emits a fluorescent signal upon cleavage by the enzyme.
Both of the above assay approaches are commonly used as high throughput assays for screening large chemical, natural product and combinatorial libraries. These assays tend to have problems related to autofluorescence of biological components due to the nature of the molecules which are screened. Many of the compounds and natural product extracts are colored or fluorescent and are present in the solution when the assay signal is monitored. This results in an assay interference which limits the detection sensitivity and the dynamic range of the assay. This interference can easily be interpreted as an inhibition of the enzyme, making it difficult to determine true positive inhibition, thus, requiring extensive follow-up assays to distinguish true positives from the false positives.
U.S. Pat. No. 5,591,591, assigned to Tropix, describes assays for the detection of proteases wherein a dioxetane compound bearing a proteolytic enzyme-specific amino acid or peptide, is added to a sample suspected of containing the protease, and the amino acid is removed by enzymatic reaction by the protease, causing the dioxetane to decompose and chemiluminescence.